Vaccine containing a peroxiredoxin and/or a β-tubulin

ABSTRACT

Vaccine compositions comprising peroxiredoxin and/or β-tubulin antigenic material, preferably of Fasciola or Dicrocoelium origin, for use in combating a parasitic infestation of helminths in a mammal, are provided. Also provided are nucleic acid sequences which encode peroxiredoxin and/or β-tubulin molecules and the amino acid sequences thereof, vectors comprising said nucleic acid sequences and cells transformed with such vectors.

The invention relates to the use of a peroxiredoxin (thiol-specificantioxidant) and/or a β-tubulin as a protective antigen against helminthparasites.

Each species of domestic animal can be parasitised by a number ofdifferent species of helminth, a process which usually causes disease.For example, the parasitic trematode Fasciola hepatica is known to beone cause of the economically important disease fascioliasis inruminants, such as cattle and sheep. The parasite enters the mammalianhost by penetrating the gut wall and spends approximately seven weeksfeeding on and burrowing through the liver mass before migrating intothe bile duct. Following infection, development of immunity in the hostcan be poor and resistance to reinfection in already infected hosts maybe only partial or non-existent. Other parasitic flukes include Fasciolagigantica and Dicrocoelium spp., Paramphistomum spp. and alsoSchistosoma spp., eg S. bovis and S. mansoni.

Problems are also caused by nematodes such as hookworms (e.g. Necator,Ancylostoma, Uncinaria and Bunostomum spp.).

Of the blood feeding nematodes the genus Haemonchus causes anaemia andweight loss and if untreated frequently leads to death. Animals infectedwith the related non-blood feeding nematode Ostertagia similarly fail tothrive and may die if untreated.

Other parasitic worms of economic importance include the various speciesof the following helminth genera:—

Trichostrongylus, Nematodirus, Dictvocaulus, Cooperia, Ascaris,Dirofilaria, Trichuris and Strongylus. In addition to domesticlivestock, companion animals and humans may also be infected, notinfrequently with fatal results and helminth infections and infestationsthus pose a problem of considerable worldwide significance.

Control of helminth parasites of grazing livestock currently reliesprimarily on the use of anthelmintic drugs combined with pasturemanagement. Such techniques are often unsatisfactory, firstly becauseanthelmintic drugs may have to be administered frequently, secondlybecause resistance against anthelmintic drugs is becoming increasinglywidespread and thirdly because appropriate pasture management is oftennot possible on some farms and even where it is, it can placeconstraints on the best use of available grazing.

Numerous attempts have been made to control helminth parasites ofdomestic animals by immunological means. With very few exceptions (e.g.the cattle lungworm, Dictyocaulus viviparus) this has not provedpossible.

A vaccine against F. hepatica has been proposed in WO90/08819 comprisinga glutathione-S-transferase from F. hepatica as antigenic material.Further vaccines against F. hepatica have been proposed in WO94/09142,WO94/28925 and PCT/GB95/02350 comprising respectively a Cathepsin L, adipeptidyl peptidase and a class of haemoproteins from F. hepatica asantigenic material.

Bennett (UK Patent No. 2169606B) extracted various antigens fromFasciola organisms by a process which separates antigens specific to thejuvenile stage from antigens present throughout the juvenile and adultstages.

Furthermore crude in vitro excretory/secretory (E/S) products can undersome circumstances confer immunity on rats (Rajasekariah et al,Parasitol. 79 (1979), p. 393-400).

It has now been found that animals vaccinated against F. hepatica usinga relatively impure haemoprotein preparation, the pure counterpart ofwhich is described in PCT/GB95/02350, produce antibodies againstperoxiredoxin and β-tubulin molecules of fluke origin. This discoveryopens up the possibility of vaccines against F. hepatica and otherhelminths based on the use of peroxiredoxin and/or β-tubulin moleculesand/or corresponding proteins produced by other helminth parasites asantigens.

Accordingly an aspect of the present invention provides a vaccinecomposition for use in combating a parasitic infestation of helminths ina mammal wherein the antigenic material comprises a peroxiredoxin and/ora β-tubulin molecule, in at least partially purified form, or anantigenic fragment or epitope, component, precursor, analogue, variantor functionally equivalent derivative thereof, together with a carrierand/or adjuvant.

The invention also provides a method of combating a parasiticinfestation of helminths in a mammal comprising administering to saidmammal a vaccine according to the invention as hereinbefore defined inan amount effective to combat said infestation.

Alternatively viewed, the invention provides for the use of themolecules as hereinbefore described in the preparation of a vaccinecomposition for combatting a parasitic infestation of helminths in amammal.

The mammal is preferably a ruminant, for example cattle or sheep, butthe vaccine and method of the invention may also find application inhumans, companion animals such as dogs and cats or in other domesticanimals.

Preferably the peroxiredoxin and/or β-tubulin molecules are derived fromflukes such as Fasciola or Dicrocoelium, in particular from the liverfluke Fasciola hepatica. Alternatively it is preferred that theperoxiredoxin and/or β-tubulin molecules should be capable ofstimulating an immune response which will be effective against Fasciolaor Dicrocoelium, in particular F. hepatica and F. gigantica, suchperoxiredoxin and/or β-tubulin molecules from other species as arecapable of conferring a cross-protective immune response thus forming aparticularly preferred aspect of the invention.

The F. hepatica peroxiredoxin and β-tubulin molecules shown hereinafterto possess cDNA sequences and predicted amino acid sequences as shown inFIGS. 2 and 4 respectively are particularly preferred for use in thevaccine and method of the invention.

The peroxiredoxin and/or β-tubulin molecules incorporated in the vaccineaccording to the invention are in at least partially purified form.Preferably the molecules of the present invention are at least 75% pureand more preferably at least 95% pure. It will be appreciated that onceperoxiredoxin and/or β-tubulin molecules of at least 95% purity havebeen obtained they can be admixed with one or more further purifiedantigenic proteins, to form a polyvalent vaccine.

According to the present invention the peroxiredoxin and/or β-tubulinmolecules incorporated in the vaccine may be in the form of antigens,antigenic fragments, epitopes, components, precursors, analogues orfunctionally-equivalent derivatives thereof.

A preferred form of polyvalent vaccine according to the invention willcontain peroxiredoxin and/or β-tubulin polypeptides as referred to abovein combination with a Cathepsin L-type antigen as described in moredetail in International Patent Application No. WO94/09142 or adipeptidyl peptidase antigen as described in more detail inInternational Patent Application No. WO94/28925 or a class ofhaemoprotein molecules as described in more detail in InternationalPatent Application No. PCT/GB95/02350. Thus the Cathepsin L and/ordipeptidyl peptidase and/or haemoproteins are preferably derived fromflukes such as Fasciola or Dicrocoelium, in particular the liver flukeF. hepatica. Such a polyvalent vaccine will, by inducing immunity in thehost species against two or more separate aspects of the invadinghelminth parasite, significantly increase the likelihood of protectionagainst the helminth and significantly reduce the chances of infestationoccurring.

Monovalent vaccines according to the invention may also have ananti-fecundity effect on helminth parasites, and this effect should bestill more marked with polyvalent vaccines.

In a preferred aspect the polyvalent vaccine comprises peroxiredoxinand/or β-tubulin polypeptides according to the present inventiontogether with a Cathepsin L1 having molecular weight of 27 kDa by sodiumdodecyl sulphate polyacrylamide gel electrophoresis as disclosed inWO94/09142 and/or a Cathepsin L2 having molecular weight of 29.5 kDa bythe same technique as disclosed in WO94/09142 and/or a dipeptidylpeptidase having molecular weight of 200 kDa by the same technique asdisclosed in WO94/28925 or one or more of a class of haemoproteins of atleast 200 kDa by gel filtration chromatography as disclosed inPCT/GB95/02350.

The vaccines according to the invention may be formulated withconventional carriers and/or adjuvants and the invention also provides aprocess for the preparation of the vaccines comprising bringing intoassociation purified peroxiredoxin and/or β-tubulin molecules ashereinbefore described and one or more adjuvants or carriers. Suitableadjuvants include aluminium hydroxide, saponin (ISCOMs), quil A and morepurified forms thereof, muramyl dipeptide, mineral and vegetable oils,DEAE dextran, nonionic block copolymers or liposomes such as Novasomes(Trade Mark of Micro Vesicular Systems Inc.), in the presence of one ormore pharmaceutically acceptable carriers or diluents. Carriers forpeptide sequences corresponding to epitopes of peroxiredoxin orβ-tubulin molecules according to the invention can be proteins such asHepatitis B core antigen multiple antigen peptide or lipopeptides suchas tripalmitoyl-S-glycerylcysteinylserylserine (P₃CSS). Suitablediluents include liquid media such as saline solution appropriate foruse as vehicles. Additional components such as preservatives may beincluded.

Administration of the vaccine to the host species may be achieved by anyof the conventional routes, e.g. orally or parenterally such as byintramuscular injection, optionally at intervals e.g. two injections ata 7-35 day interval. A suitable dose when administered by injectionmight be such as to give an amount of protein within the range 10-500μg.

According to a further aspect, the invention provides the F. hepaticaperoxiredoxin molecule or antigenic fragments, epitopes, components,precursors, analogues or variants thereof and functionally-equivalentderivatives thereof having protective antigenic activity against one ormore helminth parasites, characterised by:

(a) having at least a portion which substantially corresponds to theamino acid sequence as shown in FIG. 4;

(b) being encoded by a nucleotide sequence at least a portion of whichsubstantially corresponds to the sequence shown in FIG. 4;

While the peroxiredoxin and/or β-tubulin molecules for use in thevaccine according to the invention may be prepared by isolation from thehelminths, it may also be convenient to prepare them by recombinant DNAtechniques with the known advantages which such techniques give in termsof scaling-up of production and reproducibility. Thus the invention alsoprovides for peroxiredoxin and β-tubulin molecules as hereinbeforedescribed, produced by means of recombinant DNA techniques.

Accordingly, in one aspect, the present invention provides for nucleicacid sequences which encode the peroxiredoxin or the β-tubulin moleculesof the invention or antigenic portions thereof substantiallycorresponding to all or a portion of the nucleotide sequences as-shownin FIG. 4 for peroxiredoxin and FIG. 2 for β-tubulin or sequencesencoding helminth peroxiredoxin or β-tubulin antigens which aresubstantially homologous or which hybridise with any of said sequences.

A nucleic acid according to the invention may thus be single or doublestranded DNA, cDNA or RNA.

Variations in the peroxiredoxin or β-tubulin-encoding nucleotidesequences may occur between different strains of helminth within aspecies, between different stages of a helminth life cycle (e.g. betweenlarval and adult stages), between similar strains of differentgeographical origin, and also within the same helminth. Such variationsare included within the scope of this invention.

“Substantially homologous” as used herein includes those sequenceshaving a sequence identity of approximately 50% or more, eg. 60% ormore, and also functionally-equivalent allelic variants and relatedsequences modified by single or multiple base substitution, additionand/or deletion. By “functionally equivalent” is meant nucleic acidsequences which encode polypeptides having anti-oxidant or β-tubulinfunctionality which are similarly immunoreactive i.e. which raise hostprotective antibodies against helminths.

Nucleic acid molecules which hybridise with the sequences shown in FIGS.2 and 4 or any substantially homologous or functionally equivalentsequences as defined above are also included within the scope of theinvention. “Hybridisation” as used herein defines those sequencesbinding under non-stringent conditions (6×SSC/50% formamide at roomtemperature) and washed under conditions of low stringency (2×SSC, roomtemperature, more preferably 2×SCC, 42° C.) or conditions of higherstringency eg. 2×SSC, 65° C. (where SSC=0.15M NaCl, 0.015M sodiumcitrate, pH 7.2).

Methods for producing such derivative related sequences, for example bysite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids are well known in the art, as aremethods for determining whether the thus-modified nucleic acid hassignificant homology to the subject sequence, for example byhybridisation.

Provision of a nucleic acid molecule according to the invention thusenables recombinant peroxiredoxin or μ-tubulin or immunogenic fragmentsthereof, to be obtained in quantities heretofore unavailable, therebypermitting the development of anti-helminth vaccines.

In another aspect the present invention thus provides nucleic acidmolecules comprising one or more nucleotide sequences encoding one ormore polypeptides capable of raising protective antibodies againsthelminth parasites, which sequences incorporate one or more antigenicdeterminant-encoding regions from the peroxiredoxin or β-tubulinencoding sequences as shown in FIGS. 2 and 4.

The present invention also extends to synthetic polypeptides comprisingone or more amino acid sequences constituting a peroxiredoxin orβ-tubulin molecule or antigenic portions thereof, substantiallycorresponding to all or a portion of the nucleotide sequences as shownin FIGS. 2 and 4 or a functionally-equivalent variant thereof.

Additional aspects of the invention related to the above include vectorscontaining one or more nucleotide sequences as defined above; hostcells, for example bacteria such as E. coli or yeast cells such asSaccharomyces spp., or more preferably eukaryotic cells, transformed bysuch vectors, for example by a baculovirus vector; and processes forpreparing recombinant peroxiredoxin and β-tubulin polypeptides orantigenic fragments or epitopes thereof comprising culturing suchtransformed host cells and isolating said peroxiredoxin or β-tubulinpolypeptides or fragments or epitopes from the cultured cells.

An alternative live or inactivated vaccine formulation may comprise anattenuated or virulent virus or a host cell, e.g. a microorganism suchas a bacterium, having inserted therein a nucleic acid molecule (e.g. aDNA molecule) according to the invention for stimulation of an immuneresponse directed against polypeptides encoded by the inserted nucleicacid molecule. A bacterial vector which elicits local gut mucosalimmunity to a fluke antigen which then blocks juvenile fluke migrationis particularly preferred, notably invasive species such as Salmonellaspecies.

Additional antigenic materials may also be present in the vaccine thusgiving an enhanced protective effect against the helminth parasite inquestion or a combined protective effect against one or more additionalparasitic infestations.

A yet further aspect of the invention provides monoclonal or polyclonalantibodies capable of inducing immunity to peroxiredoxin or β-tubulinmolecules in a mammal when administered to said mammal, the antibodieshaving an affinity for the variable region of one or more furtherantibodies, said further antibodies having an affinity for saidthiol-specific antioxidant or β-tubulin molecules.

This approach, the so-called “anti-idiotype” approach, permitsformulation of a vaccine which will dispense entirely with the originalantigen and may offer even greater advantages in terms of safety,avoidance of side effects and convenience of manufacture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

PCR amplified inserts of immunoselected λ gt11 clones. Positive cloneswere amplified by PCR using universal λ forward and reverse primers.Samples of each PCR reaction were analysed by agarose gelelectrophoresis.

lanes 1, 12, 23 pGem DNA markers lane 2 clone D6 lanes 3 & 4 clones B5,D5 lanes 5-11 clones A1, A4, A5, B1, B4, B6, E3 lane 13 clone C4 lanes14-17 clone C2, D1, D7, E2 lane 18 clone D8 lanes 19 & 20 clones C1, D3lane 21 clone A8 lane 22 clone E4

FIGS. 2A and 2B

Nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequences (SEQID NOS: 2-6) of clone D6 (β-tubulin).

FIGS. 3A and 3B

Alignment of predicted amino acid sequence of clone D6 to Toxoplasmaβ-tubulin. The deduced amino acid sequence (SEQ ID NO:7) of the partialD6 sequence was aligned with that of β-tubulin from Toxoplasma gondii(SEQ ID NO:8 GenBank accession no. P10878, Nagel and Boothroyd, 1988).Boxes surround homologous regions and gaps have been introduced to givemaximum alignment. Z=not determined.

FIG. 4

Nucleotide sequence (SEQ ID NO:10) and predicted amino acid sequence of(peroxiredoxin) (clone B1).

FIGS. 5A and 5B

Alignment of predicted amino acid sequence (SEQ ID NO:10) of clone B1.The deduced amino acid sequence of clone B1 was aligned with that of ratthiol-specific antioxidant (TSA, SEQ ID NO:11, GenBank accession no.P35704), human natural killer cell enhancing factor B, (NKEF B, SEQ IDNO:12, accession no. P31945), human proliferation associated gene, (PAG,SEQ ID NO:13, accession no. X67951), human TSA (SEQ ID NO:14, Lim et al,1994, accession no. P35701), and Onchocerca volvulus TSA (SEQ ID NO:15,accession no. U09385). Boxes denote conserved residues and gaps havebeen introduced to maximise alignment. The active site cysteine residuesare indicated by arrows.

FIG. 6

Expression of clone B1 fusion protein.

A. Plate wash supernatants of wild type phage (lane 1) and clone B phage(lane 2) were subjected to reducing SDS PAGE and silver staining.

B. Following electrophoresis, SDS gels were blotted onto nitrocelluloseand probed with anti-β-galactosidase antibody. Lane 1 contains wild typephage supernatant and lane 2 contains clone B1 supernatant. Large arrowsindicate the position of β-galactosidase. Small arrows indicate theposition of B1 recombinant fusion protein.

FIG. 7

Northern blot analysis of total RNA from F. hepatica and bovine liver.

Total RNA from F. hepatica (lane 1) and from bovine liver (lane 2) waselectrophoresed in a formaldehyde agarose gel, transferred to anitrocellulose filter and probed with ³²p labelled 400 bp fragment. RNAsize markers are indicated.

FIG. 8

Protection of glutamine synthetase by liver fluke homogenate against theDTT/Fe³⁺ system. 0.5 U glutamine synthetase (GS) was incubated in thepresence of the inactivating solution (IS): 15 μM FeCl₃ and 5 mM DTT,with 0.3 mg, 0.6 mg and 0.9 mg liver fluke homogenate (LFH), for 10 minat 37° C. Reactions were then assayed for remaining glutamine synthetaseactivity.

DETAILED DESCRIPTION OF THE INVENTION:

1. Materials

The Alpha ³²p dATP was obtained from Amersham, the RNAzol™ B from AMSBiotechnology Ltd., and the X-Omat X-ray film, FX 40 liquid Fixer, LX 24developer 667 Polaroid film were all purchased from Kodak. Agarose,Anti-βgalactosidase antibody labelled with alkaline phosphatase (mouse),Apa I, 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-Gal), dNTP's,EcoR I, Hind III, isopropylthio-β-D-galactoside (IPTG), pGem DNAmarkers, pGem® vector system, Prime-a-Gene® system, Sac I, Taq DNAPolymerase, WizarD™ λ preps, Wizard™ DNA clean-up system were allpurchased from Promega. Adenine diphosphate (ADP), anti-bovine IgGconjugated to alkaline phosphatase (rabbit), diethylpyrocarbonate(DEPC), dithiothreitol (DTT), glutamine, glutamine synthetase, lysozyme,proteinase K, salmon sperm DNA all came from Sigma Chemical Company.

2. Immunoscreening of F Hepatica λ gt11 cDNA Expression Library

Preparation of λ gt11 cDNA Library

A λ gt11 cDNA Library was Prepared by the following standard method(Promega Handbook). Total RNA was isolated from mature adult flukesusing RNAzoL™. From this, mRNA was isolated by binding to an oligo dTcolumn. Double stranded cDNA was generated from the mRNA using theRiboclone® cDNA synthesis kit. EcoR I linker arms were added to thecDNA, which was then ligated to gt11 arms and packaged into λ headsusing the Packagene® system. The packaged phage was titred and thenamplified by infecting phage competent E. coli Y1090 cells (overnightculture grown in LB media with 0.2% maltose and 10 mM MgSO₄) withdilutions of the phage, incubating at room temperature for 20 min andthen plating the bacteria in top agar onto LB agar plates with 100 μgml−¹ ampicillin.

Preparation of Haemoglobin Fraction

Mature F. hepatica flukes were removed from the bile ducts of infectedlivers from condemned cattle at a local abattoir in Ireland. The flukeswere washed six times in phosphate buffered saline (PBS), pH 7.3, andthen maintained in RPMI-1640, pH 7.3, containing 2% glucose, 30 mM HEPESand 25 mg ml⁻¹ gentamycin at 37° C. for 18 hours. Following thisincubation period the culture medium was removed, centrifuged at12,000×g for 30 minutes and the supernatant (ES products) collected andstored at −20° C.

Five hundred ml of ES products were concentrated to 15 ml in an Amicon8400 Ultrafiltration unit (Danvers, Mass., USA) with a YM3 membrane(3,000 mw cut-off). The concentrated sample was centrifuged at 12,000×gfor 30 minutes and applied to a 340 ml Sephacryl S-200 columnequilibrated in 0.1M Tris-HCl, pH 7.0, at 40° C. Fractions (5 ml) werecollected after the void volume (110 ml) had been passed. The absorbanceof the eluate was monitored at 280 nm using an Atto UV Monitor. Thosefractions containing haemoprotein (yellow coloured) were pooled andconcentrated in an Amicon 8050 Ultrafiltration unit to 5 ml. Thisconcentrate was termed haemoglobin fraction (Hf).

Preparation of Sera for Immunoscreening

The cDNA library was immunologically screened using a pool of sera fromanimals vaccinated with haemoglobin fraction (Hf) as described above.The sera were obtained following three vaccinations with Hf and prior toparasite challenge. Before use the sera was pre-adsorbed to remove allantibodies reactive with E. coli proteins. This was achieved byincubating the sera with nitrocellulose discs containing bound E. coliproteins at room temperature for 6 h. This adsorption procedure wasrepeated three times. The discs were prepared by incubating the discs ina sonicated extract of E. coli cells (10×30 sec bursts, duty cycle 0.7sec) for 24 h at 4° C. and then blocking the excess sites with 1%BSA/T-PBS. Sera was incubated with discs, removed, centrifuged andstored at 40° C. until required.

Immunoscreening of λ Library

Phage competent E. coli Y1090 were infected with 1:50 dilution of phage.Following an incubation for 20 min at room temperature the cells wereplated in top agar on LB ampicillin plates and incubated at 42° C. untilplaques were visible (ca 3 h). Nitrocellulose discs which had beensoaked in 10 mM IPTG and air dried, were carefully placed on the platesand their orientation was marked by three needle stabs. The plates wereincubated for 4 h at 37° C., the discs were then carefully removed andblocked overnight in 1% BSA/T-PBS, before probing with the pre-adsorbedbovine antisera (1:500 dilution). Following washing in T-PBS boundantibody was detected using alkaline phosphatase labelled anti-bovineIgG, with NBT and BCIP as substrate. Positive plaques appeared as purplerings. These plaques were removed as an agar plug using a sterilepasteur pipette, transferred to 1 ml phage buffer (10 mM MgSO₄, 100 mMNaCl, 20 mM Tris-HCl, pH 7.4) and allowed to diffuse at 4° C. overnight.Individual phage were re-plated and the antibody screening repeated twoadditional times or until pure plaques were obtained i.e. when allplaques on a plate were reactive with the antibody.

3. Preparation of λ Lysates and Isolation of DNA

Isolated plaques were picked into 200 μl 1.0×SM buffer (0.01% gelatin, 8mM MgSO₄, 100 mM NaCl, 50 mM Tris-HCl, pH7.5) and incubated overnight at4° C. One hundred μl was used to infect competent Y1090 cells, whichwere plated as before and incubated at 42° C. until confluent lysis wasobserved (ca 5 h). Four ml 0.1×SM buffer was added to the plate andafter an overnight incubation at 4° C. the buffer was removed.Chloroform was added (0.5% final concentration) and the lysate wasstored at 4° C. until required.

4. PCR Analysis of λ DNA

Polymerase Chain Reaction (PCR) was employed to isolate and estimate thesize of the inserts from the phage library, using universal λ primers.These primers are derived from the sequence flanking the EcoR I cloningsite of the λ gt11 vector. Twenty μl of stock λlysates was added to 180μl water and boiled for 10 minutes and then 1 μl was used per 50 μl PCR.Each PCR vial consisted of the following mix:

10X Polymerase buffer  5.0 μl dNTP's (1 mM each)  5.0 μl MgCl₂ (25 mM) 6.0 μl Sterile distilled water 30.7 μl λ forward primer (50 ng μl⁻¹) 1.0 μl λ reverse primer (50 ng μl⁻¹)  1.0 μl Taq Polymerase (5 U μl⁻¹) 0.3 μl λ lysate DNA  1.0 μl

Each mix was overlaid with 70 μl mineral oil, placed in the HybaidOmnigene Thermal Cycler, and the PCR carried out as follows:

Stage 1 (Denaturation) 94° C. for 4 min Stage 2 (Denaturation) 94° C.for 30 sec (Annealing) 55° C. for 1 min (Extension) 74° C. for 1 min 30sec stage 2 was repeated for 35 cycles Stage 3 (Extension) 74° C. for 4min

25 μl of PCR reactions were analysed by agarose gel electrophoresis asdetailed in Sambrook et al (1989).

5. Sub Cloning of PCR Fragments

PCR amplified gene fragments were excised from the gel. The agarose wasdisrupted using glass beads, and the recovered DNA was purified usingthe Wizard™ DNA clean-up system (Promega). The fragments were then subcloned directly into the pGem®-T plasmid, as follows:

1 μl (25 ng) pGem®-T vector, 8 μl ligase buffer (10 mM ATP, 100 mMMgCl₂, 100 mM DTT, 300 mM tris-HCl, pH 7.8), 1 U T4 DNA ligase and 100ng insert DNA were mixed gently and the ligation was allowed to proceedovernight at 4° C.

Competent cells were prepared using one of the following methods:

(a) calcium chloride transformation A log phase culture of E. coli JM109cells was aliquoted, placed on ice for 5 min, centrifuged at 12,000×gfor 2 min and the supernatant removed. The cells were gently resuspendedwith 1 ml of cold CaCl₂ and incubated on ice for 30 min. The cells werespun again and resuspended in 0.5 ml cold CaCl₂. 10 μl ligation mix wascarefully added to 50 μl aliquots of cells and placed on ice for afurther 30 min. The cells were then heat shocked at 42° C. for 90 secand returned to ice for 2-5 min. Immediately after transformation 950 μlpre-warmed LB media was added and the cells incubated at 37° C. for 1 h.Cells were concentrated by centrifugation and spread on LB platescontaining 100 μg ml⁻¹ ampicillin, 0.5 mM IPTG and 40 μg ml⁻¹ X-Gal (forblue/white selection).

(b) electroporation

A log phase culture of E. coli XL1-blue electrocompetent cells wasconcentrated by centrifugation and aliquoted. 2.5 μl ligation reactionwas added to 300 μl cells, gently mixed and placed in 0.2 μmelectroporation cuvettes. The cells were then transformed byelectroporating under the following conditions: the pulse generator wasset at 25 μF, 2.48 kV, and 200 Ω. One pulse at these settings results ina pulse of 12.5 kV cm⁻¹ with a time constant of ca 4 sec. 1 mlpre-warmed SOC (containing 20 mM glucose) medium was added immediatelyand the cells were incubated for 1 h at 37° C., before concentrating andplating as before. Plates spread with transformed cells were incubatedovernight at 37° C.

6. Screening of Recombinant Plasmids

With X-Gal and IPTG colour screening, recombinant colonies should bewhite and colonies with no insert DNA blue. White colonies were pickedinto 2 ml LB with 100 μg ml⁻¹ ampicillin (and 15 μg ml⁻¹ tetracyclinefor XL blue cells), and incubated overnight at 37° C. Plasmid DNA from 1ml of this mini prep culture was isolated by either the boiling oralkali lysis method described by Sambrook et al (1989). The DNA wasdouble digested with the restriction enzymes Sac I and Apa I and theinserts observed on agarose gel electrophoresis.

7. Sequencing of Plasmid DNA

Purified plasmid DNA from positive clones was further cleaned up usingWizard™ λ A preps. The DNA was sent for sequence analysis to theDepartment of Biological Sciences, Durham University or BioResearchIreland, Trinity College Dublin.

8. Preparation of Fusion Protein

Sequence analysis revealed that clone B1 was a novel fluke antioxidantprotein (peroxiredoxin) which was therefore further characterised.Fusion protein from the λ B1 clone was prepared by the plate washsupernatant method. Phage competent E. coli Y1090 were infected with10,000 pfu recombinant phage and incubated for 20 min at roomtemperature, before pouring onto LB ampicillin plates in top agar. Theplates were incubated at 42° C. for 3 h (lysis almost confluent), then 5ml phage buffer containing 1 mM EDTA, 1 mM PMSF, 1 mM iodoacetamide and10 mM IPTG was added to the plates which were incubated at 37° C.overnight. The buffer was recovered and the top agar was also scrapedinto a centrifuge tube. This was vortexed for 20 sec before centrifugingat 10,000×g for 10 min at 4° C. The supernatant was removed to microfugetubes which were spun again at 12,000×g. Supernatants were stored at−20° C. until required.

The fusion protein was analysed by reducing SDS polyacrylamide gelelectrophoresis followed by silver staining, and by immunoblotting usingan anti-β galactosidase primary antibody.

9. Preparation of Radiolabelled DNA Probe

A 400 bp fragment was PCR amplified from clone B1 DNA using thefollowing consensus primers, designed from comparing the proteinsequences of the peroxiredoxin antioxidant family. These primers crossedthe regions that code for the conserved active site regions, cys 47 (VCP47) and cys 168 (VCP 168).

VCP 47 forward primer (Shem F) (SEQ ID NO:16) 5′ GAT TTY ACW TTY GTN TGTCCW ACW GAR -3′ VCP 168 reverse primer (SmTSAR) (SEQ ID NO:17) 5′ GGWCAN ACY TCW CCA TGY TC -3′

where Y=T or C, W=A or G and N=T C, A or G

The PCR product was excised from an agarose gel and cleaned as before.The fragment was labelled with Alpha ³²P by random priming using thePromega Prime-a-Genes® system. The reaction mix was as follows:

5X labelling buffer 10 μl (250 mM tris-HCl, pH 8.0, 25 mM MgCl₂,  2 μl10 mM DTT, 1 mM HEPES, pH 6.6, 26 A₂₆₀ units ml⁻¹ randomhexadeoxyribonucleotides) mixture of dCTP, dGTP, dTTP (100 mM each)acetylated BSA 10 mg ml-¹  2 μl denatured DNA probe 25 ng sterile water25 μl alpha ³²P dATP (50 μCi, 3,000 Ci mMol⁻¹)  5 μl Klenow enzyme  5 U

The reaction tube was mixed gently and incubated at room temperature for1 h. 200 μl 0.5 M EDTA was added and the reaction terminated by boilingfor 2 min. The probe was now ready for use in hybridisation reactions.

10. Isolation of RNA and Northern Blotting

a. Isolation of Adult Fluke RNA

Mature flukes were cultured overnight in RPMI-1640, pH 7.3 containing 2%glucose, 30 mM HEPES and 25 mg/l gentamycin, to allow clearing of thegut contents which could contain host cells. Approximately 10 flukes (1gram tissue) were placed in a centrifuge tube, 5 ml RNAzol™ was addedand the flukes were homogenised at top speed for 30 sec using aThyristor Regler TR50 homogeniser. One ml of chloroform was added andthe solution was shaken vigorously for 15 sec and placed on ice for 5min. After aliquoting into microfuge tubes the solution was centrifugedat 13,000×g for 15 min at 4° C. and two layers formed. The upper aqueousphase was removed to a new tube, an equal volume of isopropanol wasadded and the samples were incubated at 40° C. for 15 min (or aliquotedfor long term storage at −80° C.). They were recentrifuged for 15 min,the supernatant was removed and the RNA pellet washed with 75% ethanolbefore drying and reconstitution with 200 μl 0.1% DEPC treated water.Bovine RNA was isolated using the same procedure with 1 g fresh bovineliver as starting material. The RNA was analysed by electrophoresis onagarose gels containing formaldehyde as detailed in Sambrook et al(1989):

b. Northern Blotting

A Following electrophoresis the gel was rinsed with DEPC treated waterto remove the formaldehyde and the RNA was transferred ontonitrocellulose membrane by the capillary transfer method outlined bySambrook et al, (1989). RNA fragments are carried from the gel in a flowof buffer and deposited on the surface of the nitrocellulose. Followingtransfer, the RNA was fixed onto the membrane by baking for 2 h at 80°C. in an oven.

c. Hybridisation with Radiolabelled Probe

The nitrocellulose filter was soaked in 6×SSC (0.9 M NaCl, 90 mM sodiumcitrate pH 7.0) until thoroughly wetted and placed in a heat-sealablebag. Then, 200 ml prehybridisation solution (6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg ml denatured, fragmented salmon sperm DNA) wasadded to the bag. As much air as possible was squeezed from the bagwhich was sealed and incubated overnight at 68° C. Following incubationthe bag was opened by removing a corner and the radiolabelled probecarefully added. The resealed bag was then placed in a second sealed bagand incubated again at 68° C. for 24 h. The hybridisation solution wascarefully poured into a suitable container and the filters were removedand immediately submerged in 300 ml 2×SSC and 0.1% SDS. The filters wereincubated with gentle agitation at room temperature for 15 min. The washsolution was replaced twice and the incubation repeated. Then 0.1×SSCand 0.5% SDS was added to the filters which were further incubated at68° C. for 1 h. Filters were rinsed with 0.1×SSC to remove the SDS,blotted briefly on paper towels and wrapped in clingfilm, and thenexposed to X-ray film at −80° C. to obtain an autoradiographic image.Exposure for 24 h at 80° C. with an intensifying screen was required toobtain an image.

11. Assay of Mature Fluke Extract for Novel Antioxidant Activity

Antioxidant activity in mature liver fluke extract was measured bymonitoring its ability to inhibit the thiol/iron/oxygen mediatedinactivation of glutamine synthetase. Assays were performed inmicrotitre plates in a 100 μl reaction volume containing 0.5 U glutaminesynthetase (E. coli), in the presence or absence of inactivationsolutions and protector protein (liver fluke homogenate). Inactivationsolutions consisted of 15 μM FeCl₃ and either 5 mM DTT or 14 mM2-mercaptoethanol (final concentrations). After incubation for 10 min at37° C. remaining glutamine synthetase activity was measured by adding100 μl of γ glutamyl transferase assay mixture. This contained 0.4 mMADP, 150 mM glutamine, 10 mM potassium arsenate, 0.4 mM manganesechloride, 20 mM hydroxylammonium chloride in 50 mM imidazole-HCl, pH7.0. The reaction was incubated at 37° C. for 30 min and terminated bythe addition of 50 μl stop mixture, consisting of 55 g FeCl₃.6H₂O, 20 gtrichloroacetic acid and 21 ml concentrated HCl per liter. An absorbanceresulting from the γ glutamyl hydroxamate-Fe³⁺ complex was measured at540 nm. In the absence of “protector protein” under these conditions 70to 100% of glutamine synthetase activity was lost.

12. Immunoscreening of F. hepatica cDNA Library and Analysis of IsolatedClones by PCR and Restriction Digestion

Bovine sera from the vaccine trial was used to screen a F. hepatica cDNAlibrary constructed in λgt11 phage. The serum pool used was obtained onthe day of parasite challenge (week 11) from animals immunised withhaemoglobin fraction (Hf). These animals showed a mean level ofprotection from parasite challenge of 43.8%. This sera should containantibodies reactive with haemoglobin and any other antigens present inthe immunising fraction.

Ten plates with ca 2,000 pfu on each were used in the primary screeningwith a 1:500 dilution of pre-adsorbed sera. Thirty positive plaques werechosen and these were subjected to three or four further rounds ofscreening until all plaques on the plates were positive indicating pureclones. Lysates of positive plaques were then prepared and the DNAanalysed by PCR using λ forward and reverse primers. Of the thirtypositives selected only twenty produced PCR products; the remaining tenwere therefore disregarded. Clones were classified into groups on thebasis of the size of the PCR fragment (FIG. 1).

Group Size of PCR fragment Clones 1 ^(˜)1700 bp D6 2 ^(˜)1600 bp B5 & D53 ^(˜)1400 bp A1, A4, A5, B1, B4, B6, E3 4 ^(˜)1100 bp C4 5 ^(˜)1000 bpC2, D1, D7, E2 6  ^(˜)900 bp D8 7  ^(˜)700 bp C1 & D3 8  ^(˜)650 bp A8 9 ^(˜)550 bp E4

13. Sub Cloning of Phage Inserts

Subcloning was performed with D6 of clone Group 1 (1700 bp) and B1 ofGroup 3 (1400 bp). The A PCR products of these two clones were subcloneddirectly into the pGem®-T plasmid. White colonies were picked andscreened by double digestion with Sac I and Apa I restriction enzymes. Aclone with a 1600-1700 bp insert was isolated from D6 and a 1400-1500 bpinsert was obtained from clone B1.

14. Sequence Analysis of Clone D6

DNA from the recombinant plasmids was sequenced commercially followingpurification using Wizard™λ preps. From clone D6 a partial sequence ofca 420 bases was obtained. The deduced 141 amino acid sequence wascompared to sequences from available databases and was found to showsignificant homology with the C-terminal end of β-tubulins from variousorganisms. β-tubulins are proteins of 440-450 amino acids in length,corresponding to ca 1320 bases, therefore clone D6 of ca 1700 bases maycontain the entire F. hepatica βtubulin gene. FIG. 2 shows the alignmentof the partial D6 sequence with β-tubulin from Toxoplasma gondii. In theregion of overlap the D6 sequence shows 64% identity and 73% similaritywith the C-terminus of the protozoan tubulin.

15. Sequence Analysis of Clone B1

Clone B insert was estimated to be ca 1400 bp in length by PCRamplification using λ primers. Approximately 1200 bases of the insertwere sequenced in the 5′ to 3′ direction. This revealed a start codonATG and an open reading frame of ca 580 bases ending with the in-frametermination codon TAG. Downstream from the termination codon was stretchof about 20 adenine residues (Poly A tail), preceded by two polyadenylation sequences, AAAATAAA (SEQ ID NO:18) and AATA (SEQ ID NO:19),indicating that the clone was complete at its 3′ end. The DNA has a 5′untranslated region of ca 200 bases and a 3′ untranslated region of ca700 bases.

Clone B1 is predicted to encode a protein of 194 amino acids with acalculated molecular mass of 21,646 Da. When used to screen proteinsequence databases, the predicted amino acid sequence shows a highlysignificant identity with a novel family of antioxidant proteins, theperoxiredoxin family. Alignment of clone B1 with rat thiol specificantioxidant (TSA, GenBank accession no. P35704), human natural killercell enhancing factor B, (NKEF B, accession no. P31945), humanproliferation associated gene, (PAG, accession no. X67951), human TSA(Lim et al, 1994, accession no. P35701), and Onchocerca volvulus TSA(accession no. 009385) is shown in FIG. 3.

The protein with the highest identity is rat TSA; 57.0% and 74.6%similar. The other identities are as follows human NKEF B 56.9%, (71.5%similar) human PAG 53.8% (73.0% similar), human TSA 53.7% (71.0%similar) and Onchocerca volvulus TSA 2.0% (33.7% similar). Similaritywas observed over the entire length of the sequences and two highlyconserved domains were observed. The first of these is a sixteen aminoacid stretch at ca positions 40-60, FYPLDFTFVCPTEIIA (SEQ ID NO: 20).The second shorter domain HGEVCPA (SEQ ID NO: 21) is found at capositions 165-175.

16. Expression of Fusion Protein by Clone B1

The plate wash supernatant method was used to make fusion proteins fromclone B1 phage. The resulting supernatant and supernatant from E. coliinfected with wild type phage were analysed on reducing SDS PAGE (FIG.6A). In the wild type preparation a protein with the same molecular massas β-galactosidase was observed (lane 1). In B1 supernatants thisprotein was absent but a larger protein of molecular mass ca 160 kDa,not found in wild type, was observed (lane 2). To determine if this wasthe fusion protein, the gel was blotted onto nitrocellulose paper andprobed with anti-β-galactosidase antibody (FIG. 6B). Binding of theantibody to the large protein confirmed its identity as aβ-galactosidase fusion protein (lane 2). The antibody also bound thewild type β-galactosidase molecule (lane 1) and a number of otherproteins common to both supernatants.

17. Northern Analysis

Primers designed from the conserved domains of the antioxidant proteins(around the VCP motifs at ca positions 50 & 170), were used to amplify aDNA fragment of ca 400 bp in length. This was ³²P labelled and used toprobe both F. hepatica and bovine RNA, which were analysed on an agarosegel prior to blotting. A single transcript of ca 750 kb was found in theF. hepatica RNA (FIG. 7 lane 1). No peroxiredoxin-similar binding wasobserved in the bovine RNA (FIG. 7 lane 2).

18. Antioxidant Activity of Mature Fluke Extract

Antioxidant activity was measured as the ability of liver fluke extractto prevent the inactivation of glutamine synthetase by a mixed ironthiol inactivation system. FIG. 8 shows the inactivation of glutaminesynthetase by iron and DTT in the presence of various levels of liverfluke homogenate (LFH). Incubation of glutamine synthetase with iron andDTT results in a 70% loss of the enzymes activity. The presence of LFHprovides dose dependent protection, with 0.3 mg, 0.6 mg and 0.9 mg LFHrestoring 50%, 61% and 75% glutamine synthetase activity, respectively.

BIBLIOGRAPHY

Lim., Y. S., Cha, M. K., Kim, H. K. and Kim, I. H. 1994. Thethiol-specific antioxidant protein from human brain: gene cloning andanalysis of conserved cysteine regions. Gene 140, 279-284.

Nagel, S. D. and Boothroy, J. C. 1988. The a and b tubulins ofToxoplasma gondii are encoded by single copy genes containing multiplecopy introns. Molecular and Biochemical Parasitology 29, 261-273.

PCT/GB95/02350

Rajasekariah et al. (1979), Parasitology 79, 393-400.

Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. In MolecularCloning: A Laboratory Manua. 2nd Ed. Cold Spring Harbour LaboratoryPress.

UK Patent No. 2169606B

WO94/09142

WO94/28925

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 21 <210> SEQ ID NO 1 <211>LENGTH: 700 <212> TYPE: DNA <213> ORGANISM: Fasciola hepatica <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (2)..(700) <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (5) <223> OTHER INFORMATION: n =a, or t, or c, or g <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (9) <223> OTHER INFORMATION: n=a or t or c or g <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (11) <223> OTHER INFORMATION: n=a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(18) <223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (16) <223> OTHER INFORMATION: n= a or tor c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (26)<223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (42) <223> OTHER INFORMATION: n= a or tor c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (44)<223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (55) <223> OTHER INFORMATION: n = a ort or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (57)<223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (62) <223> OTHER INFORMATION: n= a or tor c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (67)<223> OTHER INFORMATION: n = a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (81) <223> OTHER INFORMATION: n = a ort or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(84)..(85) <223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (87) <223> OTHER INFORMATION: n=a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(89)..(90) <223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (92) <223> OTHER INFORMATION: n=a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(100) <223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (111)..(112) <223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(119) <223> OTHER INFORMATION: n = a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (121) <223> OTHER INFORMATION: n = a ort or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (126)<223> OTHER INFORMATION: n = a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (132)..(133) <223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(137) <223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (142) <223> OTHER INFORMATION: n = a ort or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (153)<223> OTHER INFORMATION: n = a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (161) <223> OTHER INFORMATION: n = a ort or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (169)<223> OTHER INFORMATION: n = a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (172) <223> OTHER INFORMATION: n = a ort or c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (177)<223> OTHER INFORMATION: n= a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (182)..(183) <223> OTHER INFORMATION:n= a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (185) <223> OTHER INFORMATION: n = a or t or c or g <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (242) <223> OTHERINFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (262) <223> OTHER INFORMATION: n = a or t or c or g<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (292) <223> OTHERINFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (325) <223> OTHER INFORMATION: n = a or t or c or g<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (574) <223> OTHERINFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (609) <223> OTHER INFORMATION: n = a or t or c or g<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (674)..(675) <223>OTHER INFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (689)..(690) <223> OTHER INFORMATION: n = a or tor c or g <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (697)<223> OTHER INFORMATION: n = a or t or c or g <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (2)..(4) <223> OTHER INFORMATION: Xaa =unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (6) <223>OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (9) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (14)..(15) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (18)..(19) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (21) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (27)..(31) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (33) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (37) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (40) <223> OTHER INFORMATION: Xaa= unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (42)<223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (44) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (46)..(47) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (51) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (54) <223> OTHER INFORMATION: Xaa= unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(56)..(57) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (59) <223> OTHER INFORMATION: Xaa =unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (61)..(62)<223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (81) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (87) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (97) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (108) <223> OTHER INFORMATION:Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(191) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (203) <223> OTHER INFORMATION: Xaa =unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (225)<223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (230) <223> OTHER INFORMATION: Xaa = unknown<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (232) <223> OTHERINFORMATION: Xaa = unknown <400> SEQUENCE: 1 c ggt ncc ant ncc ccn ancccg gta ntt aac cgg att ccc ana ntg ccc 49 Gly Xaa Xaa Xaa Pro Xaa ProVal Xaa Asn Arg Ile Pro Xaa Xaa Pro 1 5 10 15 caa aan gng ccc ncc ccngaa taa aat tcc tna ann cnc nng ntg gcc 97 Gln Xaa Xaa Pro Xaa Pro GluAsn Ser Xaa Xaa Xaa Xaa Xaa Ala 20 25 30 can tta cca acc cnn gaa acc nanaaa tnt ggg gnn cct nag ggn ccc 145 Xaa Leu Pro Thr Xaa Glu Thr Xaa LysXaa Gly Xaa Pro Xaa Xaa Pro 35 40 45 cag aac tna cac caa naa att ttn aancca ana aac nna ngg ccc cct 193 Gln Asn Xaa His Gln Xaa Ile Xaa Xaa ProXaa Asn Xaa Xaa Pro Pro 50 55 60 ttt gaa ccc act cat ggg cgc cta act taaggt ggc cgc cct gtt ccg 241 Phe Glu Pro Thr His Gly Arg Leu Thr Gly GlyArg Pro Val Pro 65 70 75 80 ngg tcg aat gtc cca tga aan aag tgg acg aacaga tgc tga atg tgc 289 Xaa Ser Asn Val Pro Xaa Lys Trp Thr Asn Arg CysMet Cys 85 90 95 agn aac aaa gaa ttc caa gct act ttg tcg aat ggn atc ccgaat aac 337 Xaa Asn Lys Glu Phe Gln Ala Thr Leu Ser Asn Xaa Ile Pro AsnAsn 100 105 110 gtg aaa act gcg gtt tgt gac att cca cct agg ggc ctt aaaatg tcg 385 Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly Leu Lys MetSer 115 120 125 gtc aca ttt gtt ggc aat agt act gcc ata caa gaa cta ttcaaa cgt 433 Val Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe LysArg 130 135 140 gtc tcc gag cag ttc acc gca atg ttc cgt cgc aaa gca ttcttg cat 481 Val Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe LeuHis 145 150 155 160 tgg tac aca ggc gaa ggt atg gac gag atg gag ttc accgag gcc gaa 529 Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe Thr GluAla Glu 165 170 175 tcg aac atg aac gat ctg gtc agt gaa tat cag caa taccaa gan gca 577 Ser Asn Met Asn Asp Leu Val Ser Glu Tyr Gln Gln Tyr GlnXaa Ala 180 185 190 acc gct gag gag gaa ggc gaa ttc cag ctg anc gcc ggcgct acc att 625 Thr Ala Glu Glu Glu Gly Glu Phe Gln Leu Xaa Ala Gly AlaThr Ile 195 200 205 acc agt tgg tct ggt gtc aaa tcc cag cat ggc gcc ggagca tcg acg 673 Thr Ser Trp Ser Gly Val Lys Ser Gln His Gly Ala Gly AlaSer Thr 210 215 220 nng ccc aat cgc cct nng tag cgn tta 700 Xaa Pro AsnArg Pro Xaa Xaa Leu 225 230 <210> SEQ ID NO 2 <211> LENGTH: 23 <212>TYPE: PRT <213> ORGANISM: Fasciola hepatica <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (2)..(4) <223> OTHER INFORMATION: Xaa =unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (6) <223>OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (9) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (14)..(15) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (18)..(19) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (21) <223> OTHERINFORMATION: Xaa = unknown <400> SEQUENCE: 2 Gly Xaa Xaa Xaa Pro Xaa ProVal Xaa Asn Arg Ile Pro Xaa Xaa Pro 1 5 10 15 Gln Xaa Xaa Pro Xaa ProGlu 20 <210> SEQ ID NO 3 <211> LENGTH: 49 <212> TYPE: PRT <213>ORGANISM: Fasciola hepatica <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (3)..(7) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (9) <223> OTHER INFORMATION: Xaa= unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (13)<223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (16) <223> OTHER INFORMATION: Xaa = unknown <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (18) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (20) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (22)..(23) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (27) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (30) <223> OTHER INFORMATION: Xaa= unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:(32)..(33) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (35) <223> OTHER INFORMATION: Xaa =unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (37)..(38)<223> OTHER INFORMATION: Xaa = unknown <400> SEQUENCE: 3 Asn Ser Xaa XaaXaa Xaa Xaa Ala Xaa Leu Pro Thr Xaa Glu Thr Xaa 1 5 10 15 Lys Xaa GlyXaa Pro Xaa Xaa Pro Gln Asn Xaa His Gln Xaa Ile Xaa 20 25 30 Xaa Pro XaaAsn Xaa Xaa Pro Pro Phe Glu Pro Thr His Gly Arg Leu 35 40 45 Thr <210>SEQ ID NO 4 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Fasciolahepatica <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (7) <223>OTHER INFORMATION: Xaa = unknown <400> SEQUENCE: 4 Gly Gly Arg Pro ValPro Xaa Ser Asn Val Pro 1 5 10 <210> SEQ ID NO 5 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Fasciola hepatica <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (1) <223> OTHER INFORMATION: Xaa =unknown <400> SEQUENCE: 5 Xaa Lys Trp Thr Asn Arg Cys 1 5 <210> SEQ IDNO 6 <211> LENGTH: 136 <212> TYPE: PRT <213> ORGANISM: Fasciola hepatica<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (3) <223> OTHERINFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (14) <223> OTHER INFORMATION: Xaa = unknown <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (97) <223> OTHER INFORMATION: Xaa= unknown <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (109)<223> OTHER INFORMATION: Xaa = unknown <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (131) <223> OTHER INFORMATION: Xaa = unknown<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (136) <223> OTHERINFORMATION: Xaa = unknown <400> SEQUENCE: 6 Met Cys Xaa Asn Lys Glu PheGln Ala Thr Leu Ser Asn Xaa Ile Pro 1 5 10 15 Asn Asn Val Lys Thr AlaVal Cys Asp Ile Pro Pro Arg Gly Leu Lys 20 25 30 Met Ser Val Thr Phe ValGly Asn Ser Thr Ala Ile Gln Glu Leu Phe 35 40 45 Lys Arg Val Ser Glu GlnPhe Thr Ala Met Phe Arg Arg Lys Ala Phe 50 55 60 Leu His Trp Tyr Thr GlyGlu Gly Met Asp Glu Met Glu Phe Thr Glu 65 70 75 80 Ala Glu Ser Asn MetAsn Asp Leu Val Ser Glu Tyr Gln Gln Tyr Gln 85 90 95 Xaa Ala Thr Ala GluGlu Glu Gly Glu Phe Gln Leu Xaa Ala Gly Ala 100 105 110 Thr Ile Thr SerTrp Ser Gly Val Lys Ser Gln His Gly Ala Gly Ala 115 120 125 Ser Thr XaaPro Asn Arg Pro Xaa 130 135 <210> SEQ ID NO 7 <211> LENGTH: 141 <212>TYPE: PRT <213> ORGANISM: Fasciola hepatica <400> SEQUENCE: 7 Arg CysMet Cys Glx Asn Lys Glu Phe Gln Ala Thr Leu Ser Asn Glx 1 5 10 15 IlePro Asn Asn Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly 20 25 30 LeuLys Met Ser Val Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu 35 40 45 LeuPhe Lys Arg Val Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys 50 55 60 AlaPhe Leu His Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe 65 70 75 80Thr Glu Ala Glu Ser Asn Met Asn Asp Leu Val Ser Glu Tyr Gln Gln 85 90 95Tyr Gln Glx Ala Thr Ala Glu Glu Glu Gly Glu Phe Gln Leu Glx Ala 100 105110 Gly Ala Thr Ile Thr Ser Trp Ser Gly Val Lys Ser Gln His Gly Ala 115120 125 Gly Ala Ser Thr Glx Pro Asn Arg Pro Glx Asx Glx Leu 130 135 140<210> SEQ ID NO 8 <211> LENGTH: 449 <212> TYPE: PRT <213> ORGANISM:Toxoplasma gondii <300> PUBLICATION INFORMATION: <301> AUTHORS: Nagel,S. D. Boothroyd, J. C. <302> TITLE: The a and b tubulins of Taxoplasmagondii are encoded by single copy genes containing multiple copyintrons. <303> JOURNAL: Mol. Biochem. Parasitol. <304> VOLUME: 29 <306>PAGES: 261-273 <307> DATE: 1988 <300> PUBLICATION INFORMATION: <302>TITLE: GenBank Acession no. P10878 <400> SEQUENCE: 8 Met Arg Glu Ile ValHis Val Gln Gly Gly Gln Cys Gly Asn Gln Ile 1 5 10 15 Gly Ala Lys PheTrp Glu Val Ile Ser Asp Glu His Gly Ile Asp Pro 20 25 30 Thr Gly Thr TyrCys Gly Asp Ser Asp Leu Gln Leu Glu Arg Ile Asn 35 40 45 Val Phe Tyr AsnGlu Ala Thr Gly Gly Arg Phe Val Pro Arg Ala Ile 50 55 60 Leu Met Asp LeuGlu Pro Gly Thr Met Asp Ser Val Arg Ala Gly Pro 65 70 75 80 Phe Gly GlnLeu Phe Arg Pro Asp Asn Phe Val Phe Gly Gln Thr Gly 85 90 95 Ala Gly AsnAsn Trp Ala Lys Gly His Tyr Thr Glu Gly Ala Glu Leu 100 105 110 Ile AspSer Val Leu Asp Val Val Arg Lys Glu Ala Glu Gly Cys Asp 115 120 125 CysLeu Gln Gly Phe Gln Ile Thr His Ser Leu Gly Gly Gly Thr Gly 130 135 140Ser Gly Met Gly Thr Leu Leu Ile Ser Lys Val Arg Glu Glu Tyr Pro 145 150155 160 Asp Arg Ile Met Glu Thr Phe Ser Val Phe Pro Ser Pro Lys Val Ser165 170 175 Asp Thr Val Val Glu Pro Tyr Asn Ala Thr Leu Ser Val His GlnLeu 180 185 190 Val Glu Asn Ala Asp Glu Val Gln Val Ile Asp Asn Glu AlaLeu Tyr 195 200 205 Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr Thr Pro ThrTyr Gly Asp 210 215 220 Leu Asn His Leu Val Ser Ala Ala Met Ser Gly ValThr Cys Cys Leu 225 230 235 240 Arg Phe Pro Gly Gln Leu Asn Ser Asp LeuArg Lys Leu Ala Val Asn 245 250 255 Leu Val Pro Phe Pro Arg Leu His PhePhe Leu Ile Gly Phe Ala Pro 260 265 270 Leu Thr Ser Arg Gly Ser Gln GlnTyr Arg Ala Leu Ser Val Pro Glu 275 280 285 Leu Thr Gln Gln Met Phe AspAla Lys Asn Met Met Cys Ala Ser Asp 290 295 300 Pro Arg His Gly Arg TyrLeu Thr Ala Ser Ala Met Phe Arg Gly Arg 305 310 315 320 Met Ser Thr LysGlu Val Asp Glu Gln Met Leu Asn Val Gln Asn Lys 325 330 335 Asn Ser SerTyr Phe Val Glu Trp Ile Pro Asn Asn Met Lys Ser Ser 340 345 350 Val CysAsp Ile Pro Pro Lys Gly Leu Lys Met Ser Val Thr Phe Val 355 360 365 GlyAsn Ser Thr Ala Ile Gln Glu Met Phe Lys Arg Val Ser Asp Gln 370 375 380Phe Thr Ala Met Phe Arg Arg Lys Ala Phe Leu His Trp Tyr Thr Gly 385 390395 400 Glu Gly Met Asp Glu Met Glu Phe Thr Glu Ala Glu Ser Asn Met Asn405 410 415 Asp Leu Val Ser Glu Tyr Gln Gln Tyr Gln Asp Ala Thr Ala GluGlu 420 425 430 Glu Gly Glu Phe Asp Glu Glu Glu Gly Glu Met Gly Ala GluGlu Gly 435 440 445 Ala <210> SEQ ID NO 9 <211> LENGTH: 1163 <212> TYPE:DNA <213> ORGANISM: Fasciola hepatica <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (179)..(763) <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (90) <223> OTHER INFORMATION: n = a or t or c or g <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (856) <223> OTHERINFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (955) <223> OTHER INFORMATION: n = a or t or c or g<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (1026) <223> OTHERINFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (1043) <223> OTHER INFORMATION: n = a or t or c or g<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (1046) <223> OTHERINFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (1050) <223> OTHER INFORMATION: n = a or t or c or g<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (1053) <223> OTHERINFORMATION: n = a or t or c or g <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (1056) <223> OTHER INFORMATION: n = a or t or c or g<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (1122) <223> OTHERINFORMATION: n = a or t or c or g <400> SEQUENCE: 9 tcgctcactatagggcgaat tgggcccgac gtcgcatgcc cccggccgcc atggccgcgg 60 gattggtggcgacgactcct ggagcccgtn agtatcagcg gaattccggt gtgatcgcaa 120 tcagtgctctccggggcgcc atccacttcc ccactctcat ccgcatttcc aaagaccg 178 atg ttg cag cctaat atg ccc gcc ccg aat ttt tct gga cag gcg gta 226 Met Leu Gln Pro AsnMet Pro Ala Pro Asn Phe Ser Gly Gln Ala Val 1 5 10 15 gtg ggc aag gagttc gaa acc atc agt tta tca gac tac aag ggc aaa 274 Val Gly Lys Glu PheGlu Thr Ile Ser Leu Ser Asp Tyr Lys Gly Lys 20 25 30 tgg gtg att ctc gccttc tat cca ctt gat ttc acg ttc gtg tgt cca 322 Trp Val Ile Leu Ala PheTyr Pro Leu Asp Phe Thr Phe Val Cys Pro 35 40 45 acg gaa ata atc gcg atcagt gat cag atg gag cag ttc gca caa cgt 370 Thr Glu Ile Ile Ala Ile SerAsp Gln Met Glu Gln Phe Ala Gln Arg 50 55 60 aac tgc gcc gtc atc ttc tgctct act gac tcg gtt tat tcg cat ctg 418 Asn Cys Ala Val Ile Phe Cys SerThr Asp Ser Val Tyr Ser His Leu 65 70 75 80 caa tgg acc aaa atg gat cgtaag gtt ggc ggt ata ggc cag ctg aac 466 Gln Trp Thr Lys Met Asp Arg LysVal Gly Gly Ile Gly Gln Leu Asn 85 90 95 ttc ccg ctg ctg gca gac aag aatatg tct gtc tct cgc gcc ttt ggt 514 Phe Pro Leu Leu Ala Asp Lys Asn MetSer Val Ser Arg Ala Phe Gly 100 105 110 gtt ctg gat gag gaa cag ggt aatacc tac cgt ggc aat ttc ctc atc 562 Val Leu Asp Glu Glu Gln Gly Asn ThrTyr Arg Gly Asn Phe Leu Ile 115 120 125 gat ccc aag ggg gtc ctg cgc cagatc acg gtg aat gac gac ccg gtg 610 Asp Pro Lys Gly Val Leu Arg Gln IleThr Val Asn Asp Asp Pro Val 130 135 140 ggc cgt tcc gtt gaa gaa gcc ttgcgt ctg ctc gat gca ttc ata ttc 658 Gly Arg Ser Val Glu Glu Ala Leu ArgLeu Leu Asp Ala Phe Ile Phe 145 150 155 160 cac gag gag cat gga gag gtctgc ccg gcg aac tgg aag cct aaa agc 706 His Glu Glu His Gly Glu Val CysPro Ala Asn Trp Lys Pro Lys Ser 165 170 175 aag acc atc gtg cct act ccggat gga tcc aaa gca tat ttc tcc tca 754 Lys Thr Ile Val Pro Thr Pro AspGly Ser Lys Ala Tyr Phe Ser Ser 180 185 190 gcc aac tag tgaacaagggtgcttaatcc cggctctgtg tttcgtttct 803 Ala Asn 195 ggtttaaaat aaattagataatacggtgca aaaaaaaaaa aaaaaacgga atnccggtac 863 ggtaacagtt cccaagcgcaacagtatgat gagaatccaa ctgattatcg tcttggaatc 923 gctcattggt ttcgcaaccagttttcgact gnaggcaacc gcattcaagg attgtggctc 983 gcaacttgcc gaattgatgaatgtgactgt gaaaccatgt ganactactc tgtgtagtgn 1043 gtntcgnggn ganaacgcccaactggaaat cacttcccga acaaaggaag ttggcaagtc 1103 ttgaaagcag tcggccgtncaatagtcgga cgtgtttctg cccatccccc tggatgacta 1163 <210> SEQ ID NO 10<211> LENGTH: 194 <212> TYPE: PRT <213> ORGANISM: Fasciola hepatica<400> SEQUENCE: 10 Met Leu Gln Pro Asn Met Pro Ala Pro Asn Phe Ser GlyGln Ala Val 1 5 10 15 Val Gly Lys Glu Phe Glu Thr Ile Ser Leu Ser AspTyr Lys Gly Lys 20 25 30 Trp Val Ile Leu Ala Phe Tyr Pro Leu Asp Phe ThrPhe Val Cys Pro 35 40 45 Thr Glu Ile Ile Ala Ile Ser Asp Gln Met Glu GlnPhe Ala Gln Arg 50 55 60 Asn Cys Ala Val Ile Phe Cys Ser Thr Asp Ser ValTyr Ser His Leu 65 70 75 80 Gln Trp Thr Lys Met Asp Arg Lys Val Gly GlyIle Gly Gln Leu Asn 85 90 95 Phe Pro Leu Leu Ala Asp Lys Asn Met Ser ValSer Arg Ala Phe Gly 100 105 110 Val Leu Asp Glu Glu Gln Gly Asn Thr TyrArg Gly Asn Phe Leu Ile 115 120 125 Asp Pro Lys Gly Val Leu Arg Gln IleThr Val Asn Asp Asp Pro Val 130 135 140 Gly Arg Ser Val Glu Glu Ala LeuArg Leu Leu Asp Ala Phe Ile Phe 145 150 155 160 His Glu Glu His Gly GluVal Cys Pro Ala Asn Trp Lys Pro Lys Ser 165 170 175 Lys Thr Ile Val ProThr Pro Asp Gly Ser Lys Ala Tyr Phe Ser Ser 180 185 190 Ala Asn <210>SEQ ID NO 11 <211> LENGTH: 198 <212> TYPE: PRT <213> ORGANISM: Rattusrattus <300> PUBLICATION INFORMATION: <302> TITLE: GenBank accession no.P35704 <400> SEQUENCE: 11 Met Ala Ser Gly Asn Ala His Ile Gly Lys ProAla Pro Asp Phe Thr 1 5 10 15 Gly Thr Ala Val Val Asp Gly Ala Phe LysGlu Ile Lys Leu Ser Asp 20 25 30 Tyr Arg Gly Lys Tyr Val Val Leu Phe PheTyr Pro Leu Asp Phe Thr 35 40 45 Phe Val Cys Pro Thr Glu Ile Ile Ala PheSer Asp His Ala Glu Asp 50 55 60 Phe Arg Lys Leu Gly Cys Glu Val Leu GlyVal Ser Val Asp Ser Gln 65 70 75 80 Phe Thr His Leu Ala Trp Ile Asn ThrPro Arg Lys Glu Gly Gly Leu 85 90 95 Gly Pro Leu Asn Ile Pro Leu Leu AlaAsp Val Thr Lys Ser Leu Ser 100 105 110 Gln Asn Tyr Gly Val Leu Lys AsnAsp Glu Gly Ile Ala Tyr Arg Gly 115 120 125 Leu Phe Ile Ile Asp Ala LysGly Val Leu Arg Gln Ile Thr Val Asn 130 135 140 Asp Leu Pro Val Gly ArgSer Val Asp Glu Ala Leu Arg Leu Val Gln 145 150 155 160 Ala Phe Gln TyrThr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp 165 170 175 Lys Pro GlySer Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu 180 185 190 Tyr PheSer Lys His Asn 195 <210> SEQ ID NO 12 <211> LENGTH: 198 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION: <302> TITLE:GenBank accession no. P31945 <400> SEQUENCE: 12 Met Ala Ser Gly Asn AlaArg Ile Gly Lys Pro Ala Pro Asp Phe Lys 1 5 10 15 Ala Thr Ala Val ValAsp Gly Ala Phe Lys Glu Val Lys Leu Ser Asp 20 25 30 Tyr Lys Gly Lys TyrVal Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr 35 40 45 Phe Val Cys Pro ThrGlu Ile Ile Ala Phe Ser Asn Arg Ala Glu Asp 50 55 60 Phe Arg Lys Leu GluVal Leu Gly Val Ser Val Asp Ser Gln Phe Asn 65 70 75 80 His Leu Ala TrpIle Asn Thr Pro Arg Lys Glu Gly Gly Leu Gly Pro 85 90 95 Leu Asn Ile ProLeu Leu Gly Asp Val Thr Arg Arg Leu Ser Glu Asp 100 105 110 Tyr Gly ValLeu Lys Thr Asp Glu Gly Ile Ala Tyr Arg Gly Leu Phe 115 120 125 Ile IleAsp Gly Lys Gly Val Leu Arg Gln Ile Thr Val Asn Asp Leu 130 135 140 ProVal Gly Arg Ser Val Asp Glu Ala Leu Arg Leu Val Gln Ala Phe 145 150 155160 Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp Lys Pro 165170 175 Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu Tyr Phe180 185 190 Ser Lys His Asn Asn Glu 195 <210> SEQ ID NO 13 <211> LENGTH:198 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <300> PUBLICATIONINFORMATION: <302> TITLE: GenBank accession no. X67951 <400> SEQUENCE:13 Met Ser Ser Gly Asn Ala Lys Ile Gly His Pro Ala Pro Asn Phe Lys 1 510 15 Ala Thr Ala Val Met Pro Asp Gly Phe Lys Asp Ile Ser Leu Ser Asp 2025 30 Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Phe Thr 3540 45 Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Glu Glu 5055 60 Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Ser His 6570 75 80 Phe Cys His Leu Ala Trp Val Asn Thr Pro Lys Lys Gln Gly Gly Leu85 90 95 Gly Pro Met Asn Ile Pro Leu Val Ser Asp Pro Lys Arg Thr Ile Ala100 105 110 Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe ArgGly 115 120 125 Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile ThrVal Asn 130 135 140 Asp Leu Pro Val Gly Arg Ser Val Asp Glu Thr Leu ArgLeu Val Gln 145 150 155 160 Ala Phe Gln Phe Thr Asp Lys His Gly Glu ValCys Pro Ala Gly Trp 165 170 175 Lys Pro Gly Ser Asp Thr Ile Lys Pro AspVal Gln Lys Ser Lys Glu 180 185 190 Tyr Phe Ser Lys Gln Lys 195 <210>SEQ ID NO 14 <211> LENGTH: 198 <212> TYPE: PRT <213> ORGANISM: Homosapiens <300> PUBLICATION INFORMATION: <301> AUTHORS: Lim, Y. S. Cha, M.K. Kim, H. K. <302> TITLE: The thiol-specific antioxidant protein fromhuman brain: gene cloning and analysis of conserved cysteine regions.<303> JOURNAL: Gene <304> VOLUME: 140 <306> PAGES: 279-284 <307> DATE:1994 <300> PUBLICATION INFORMATION: <302> TITLE: GenBank accession no.P35701 <400> SEQUENCE: 14 Met Ala Ser Gly Asn Ala Arg Ile Gly Lys ProAla Pro Asp Phe Lys 1 5 10 15 Ala Thr Ala Val Val Asp Gly Ala Phe LysGlu Val Lys Leu Ser Asp 20 25 30 Tyr Lys Gly Lys Tyr Val Val Leu Phe PheTyr Pro Leu Asp Phe Thr 35 40 45 Phe Val Cys Pro Thr Glu Ile Ile Ala PheThr Thr Val Lys Arg Thr 50 55 60 Ser Ala Lys Leu Gly Cys Glu Val Leu GlyVal Ser Val Asp Ser Gln 65 70 75 80 Phe Thr His Leu Ala Trp Ile Asn ThrPro Arg Lys Glu Gly Gly Leu 85 90 95 Gly Pro Leu Asn Ile Pro Leu Leu AlaAsp Val Thr Arg Arg Leu Ser 100 105 110 Glu Asp Tyr Gly Val Leu Lys AsnAsp Glu Gly Ile Ala Tyr Arg Gly 115 120 125 Leu Phe Ile Ile Asp Gly LysGly Val Leu Arg Gln Ile Thr Val Asn 130 135 140 Asp Leu Pro Val Gly ArgSer Val Asp Glu Ala Leu Arg Leu Val Gln 145 150 155 160 Ala Phe Gln TyrThr Asp Glu His Gly Glu Val Cys Pro Ala Ala Trp 165 170 175 Lys Pro GlyArg Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu 180 185 190 Tyr PheSer Lys His Asn 195 <210> SEQ ID NO 15 <211> LENGTH: 161 <212> TYPE: PRT<213> ORGANISM: Onchocerca volvulus <300> PUBLICATION INFORMATION: <302>TITLE: GenBank accession no. U09385 <400> SEQUENCE: 15 Glu Phe Lys LysArg Asn Val Lys Leu Ile Gly Leu Ser Cys Asp Ser 1 5 10 15 Ala Asp SerHis Ser Lys Trp Ala Asp Asp Ile Leu Ala Leu Tyr Lys 20 25 30 Met Lys CysVal Gly Cys Asp Ser Glu Lys Lys Leu Pro Tyr Pro Ile 35 40 45 Ile Ala AspGlu Asp Arg Ser Leu Ala Thr Glu Leu Gly Met Met Asp 50 55 60 Pro Asp GluArg Asp Glu Lys Gly Asn Thr Leu Thr Ala Arg Cys Val 65 70 75 80 Phe IleIle Gly Ser Asp Lys Thr Leu Lys Leu Ser Ile Leu Tyr Pro 85 90 95 Ala ThrThr Gly Arg Asn Phe Asp Glu Ile Leu Arg Val Val Asp Ser 100 105 110 LeuGln Leu Thr Ala Val Lys Leu Val Ala Thr Pro Val Asp Trp Lys 115 120 125Asp Gly Asp Asp Cys Val Val Leu Pro Thr Ile Asp Asp Asn Glu Ala 130 135140 Lys Lys Leu Phe Gly Glu Lys Ile His Thr Ile Asp Leu Pro Ser Gly 145150 155 160 Lys <210> SEQ ID NO 16 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer coding for theconserved active site region, Cys 47. <220> FEATURE: <221> NAME/KEY:misc_structure <222> LOCATION: (6) <223> OTHER INFORMATION: y = t or c<220> FEATURE: <221> NAME/KEY: misc_structure <222> LOCATION: (9) <223>OTHER INFORMATION: r = a or g <220> FEATURE: <221> NAME/KEY:misc_structure <222> LOCATION: (21) <223> OTHER INFORMATION: r = a or g<220> FEATURE: <221> NAME/KEY: misc_structure <222> LOCATION: (24) <223>OTHER INFORMATION: r = a or g <220> FEATURE: <221> NAME/KEY:misc_structure <222> LOCATION: (15) <223> OTHER INFORMATION: n = a or gor t or c <220> FEATURE: <221> NAME/KEY: misc_structure <222> LOCATION:(12) <223> OTHER INFORMATION: y = t or c <400> SEQUENCE: 16 gatttyacrttygtntgtcc racrga 26 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer coding for theconserved active site region, Cys 168 <220> FEATURE: <221> NAME/KEY:misc_structure <222> LOCATION: (3) <223> OTHER INFORMATION: w = a or g<220> FEATURE: <221> NAME/KEY: misc_structure <222> LOCATION: (9) <223>OTHER INFORMATION: y = t or c <220> FEATURE: <221> NAME/KEY:misc_structure <222> LOCATION: (12) <223> OTHER INFORMATION: w = a or g<220> FEATURE: <221> NAME/KEY: misc_structure <222> LOCATION: (18) <223>OTHER INFORMATION: y = t or c <220> FEATURE: <221> NAME/KEY:misc_structure <222> LOCATION: (6) <223> OTHER INFORMATION: n = a or gor t or c <400> SEQUENCE: 17 ggwcanacyt cwccatgytc 20 <210> SEQ ID NO 18<211> LENGTH: 8 <212> TYPE: DNA <213> ORGANISM: Fasciola hepatica <400>SEQUENCE: 18 aaaataaa 8 <210> SEQ ID NO 19 <211> LENGTH: 4 <212> TYPE:DNA <213> ORGANISM: Fasciola hepatica <400> SEQUENCE: 19 aata 4 <210>SEQ ID NO 20 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Fasciolahepatica <400> SEQUENCE: 20 Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys ProThr Glu Ile Ile Ala 1 5 10 15 <210> SEQ ID NO 21 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Fasciola hepatica <400> SEQUENCE: 21 His GlyGlu Val Cys Pro Ala 1 5

What is claimed is:
 1. An isolated and purified protein comprising theamino acid sequence shown in SEQ ID NO:10.
 2. A composition comprisingan isolated protein having the amino acid sequence shown in SEQ ID NO:10and at least one isolated protein having an amino acid sequence as shownin SEQ ID Nos: 2, 3, 4, 5 or 6.